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1.
Phys Chem Chem Phys ; 23(24): 13745-13751, 2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34159970

RESUMO

DNA damage leads to stalled or collapsed replication forks. Replication restart primosomes re-initiate DNA synthesis at these stalled or collapsed DNA replication forks, which is important for bacterial survival. Primosomal protein PriA specifically recognizes the DNA fork structure and recruits other primosomal proteins to load the replicative helicase, in order to re-establish the replication fork. PriA binding on DNA is the first step to restart replication forks for proper DNA repair. Using a single-molecule fluorescence colocalization experiment, we measured the thermodynamic and real-time kinetic properties of fluorescence-labeled Gram-positive bacteria Geobacillus stearothermophilus PriA binding on DNA forks. We showed that PriA preferentially binds to a DNA fork structure with a fully duplexed leading strand at sub-nanomolar affinity (Kd = 268 ± 99 pM). PriA binds dynamically, and its association and dissociation rate constants can be determined using the appearance and disappearance of the fluorescence signal. In addition, we showed that PriA binds to DNA forks as a monomer using photobleaching step counting. This information offers a molecular basis essential for understanding the mechanism of replication restart.


Assuntos
Proteínas de Bactérias/química , DNA Bacteriano/química , Proteínas de Ligação a DNA/química , Geobacillus stearothermophilus/química , Sítios de Ligação , Replicação do DNA , Imagem Óptica
2.
Nucleic Acids Res ; 49(12): 6982-6995, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34161591

RESUMO

REP, diverse palindromic DNA sequences found at high copy number in many bacterial genomes, have been attributed important roles in cell physiology but their dissemination mechanisms are poorly understood. They might represent non-autonomous transposable elements mobilizable by TnpAREP, the first prokaryotic domesticated transposase associated with REP. TnpAREP, fundamentally different from classical transposases, are members of the HuH superfamily and closely related to the transposases of the IS200/IS605 family. We previously showed that Escherichia coli TnpAREP processes cognate single stranded REP in vitro and that this activity requires the integrity of the REP structure, in particular imperfect palindromes interrupted by a bulge and preceded by a conserved DNA motif. A second group of REPs rather carry perfect palindromes, raising questions about how the latter are recognized by their cognate TnpAREP. To get insight into the importance of REP structural and sequence determinants in these two groups, we developed an in vitro activity assay coupled to a mutational analysis for three different TnpAREP/REP duos via a SELEX approach. We also tackled the question of how the cleavage site is selected. This study revealed that two TnpAREP groups have co-evolved with their cognate REPs and use different strategies to recognize their REP substrates.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Bacteriano/química , Genoma Bacteriano , Sequências Repetidas Invertidas , Transposases/metabolismo , Escherichia coli/genética , Marinomonas/genética , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Técnica de Seleção de Aptâmeros , Stenotrophomonas maltophilia/genética
3.
Nat Commun ; 12(1): 3967, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34172734

RESUMO

Bottom-up and top-down approaches to synthetic biology each employ distinct methodologies with the common aim to harness living systems. Here, we realize a strategic merger of both approaches to convert light into proton gradients for the actuation of synthetic cellular systems. We genetically engineer E. coli to overexpress the light-driven inward-directed proton pump xenorhodopsin and encapsulate them in artificial cell-sized compartments. Exposing the compartments to light-dark cycles, we reversibly switch the pH by almost one pH unit and employ these pH gradients to trigger the attachment of DNA structures to the compartment periphery. For this purpose, a DNA triplex motif serves as a nanomechanical switch responding to the pH-trigger of the E. coli. When DNA origami plates are modified with the pH-sensitive triplex motif, the proton-pumping E. coli can trigger their attachment to giant unilamellar lipid vesicles (GUVs) upon illumination. A DNA cortex is formed upon DNA origami polymerization, which sculpts and deforms the GUVs. We foresee that the combination of bottom-up and top down approaches is an efficient way to engineer synthetic cells.


Assuntos
DNA Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Genética/métodos , Prótons , DNA Bacteriano/química , Concentração de Íons de Hidrogênio , Luz , Microrganismos Geneticamente Modificados , Bombas de Próton/genética , Bombas de Próton/metabolismo , Rodopsina/genética , Rodopsina/metabolismo
4.
Nat Commun ; 12(1): 3436, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103525

RESUMO

Clostridioides difficile infections are an urgent medical problem. The newly discovered C. difficile adenine methyltransferase A (CamA) is specified by all C. difficile genomes sequenced to date (>300), but is rare among other bacteria. CamA is an orphan methyltransferase, unassociated with a restriction endonuclease. CamA-mediated methylation at CAAAAA is required for normal sporulation, biofilm formation, and intestinal colonization by C. difficile. We characterized CamA kinetic parameters, and determined its structure bound to DNA containing the recognition sequence. CamA contains an N-terminal domain for catalyzing methyl transfer, and a C-terminal DNA recognition domain. Major and minor groove DNA contacts in the recognition site involve base-specific hydrogen bonds, van der Waals contacts and the Watson-Crick pairing of a rearranged A:T base pair. These provide sufficient sequence discrimination to ensure high specificity. Finally, the surprisingly weak binding of the methyl donor S-adenosyl-L-methionine (SAM) might provide avenues for inhibiting CamA activity using SAM analogs.


Assuntos
Adenina/metabolismo , Clostridioides/enzimologia , DNA Bacteriano/química , Conformação de Ácido Nucleico , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Pareamento de Bases , Sequência de Bases , Coenzimas/metabolismo , Modelos Moleculares , Motivos de Nucleotídeos , S-Adenosil-Homocisteína/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , Especificidade da Espécie , Especificidade por Substrato
5.
Nat Commun ; 12(1): 2702, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976201

RESUMO

Bacterial RNA polymerase (RNAP) holoenzyme initiates transcription by recognizing the conserved -35 and -10 promoter elements that are optimally separated by a 17-bp spacer. The MerR family of transcriptional regulators activate suboptimal 19-20 bp spacer promoters in response to myriad cellular signals, ranging from heavy metals to drug-like compounds. The regulation of transcription by MerR family regulators is not fully understood. Here we report one crystal structure of a multidrug-sensing MerR family regulator EcmrR and nine cryo-electron microscopy structures that capture the EcmrR-dependent transcription process from promoter opening to initial transcription to RNA elongation. These structures reveal that EcmrR is a dual ligand-binding factor that reshapes the suboptimal 19-bp spacer DNA to enable optimal promoter recognition, sustains promoter remodeling to stabilize initial transcribing complexes, and finally dissociates from the promoter to reverse DNA remodeling and facilitate the transition to elongation. Our findings yield a comprehensive model for transcription regulation by MerR family factors and provide insights into the transition from transcription initiation to elongation.


Assuntos
Proteínas de Bactérias/química , DNA Bacteriano/química , Proteínas de Ligação a DNA/química , RNA Polimerases Dirigidas por DNA/química , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Iniciação da Transcrição Genética , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Elongação da Transcrição Genética
6.
Nat Commun ; 12(1): 2967, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016970

RESUMO

Allostery is a pervasive principle to regulate protein function. Growing evidence suggests that also DNA is capable of transmitting allosteric signals. Yet, whether and how DNA-mediated allostery plays a regulatory role in gene expression remained unclear. Here, we show that DNA indeed transmits allosteric signals over long distances to boost the binding cooperativity of transcription factors. Phenotype switching in Bacillus subtilis requires an all-or-none promoter binding of multiple ComK proteins. We use single-molecule FRET to demonstrate that ComK-binding at one promoter site increases affinity at a distant site. Cryo-EM structures of the complex between ComK and its promoter demonstrate that this coupling is due to mechanical forces that alter DNA curvature. Modifications of the spacer between sites tune cooperativity and show how to control allostery, which allows a fine-tuning of the dynamic properties of genetic circuits.


Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , DNA Bacteriano/química , Regulação Bacteriana da Expressão Gênica , Fatores de Transcrição/genética , Regulação Alostérica/genética , Sítios de Ligação/genética , DNA Bacteriano/genética , Redes Reguladoras de Genes , Conformação de Ácido Nucleico , Fenótipo , Regiões Promotoras Genéticas/genética
7.
Nat Commun ; 12(1): 2641, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976175

RESUMO

The mechanism of DNA synthesis has been inferred from static structures, but the absence of temporal information raises longstanding questions about the order of events in one of life's most central processes. Here we follow the reaction pathway of a replicative DNA polymerase using time-resolved X-ray crystallography to elucidate the order and transition between intermediates. In contrast to the canonical model, the structural changes observed in the time-lapsed images reveal a catalytic cycle in which translocation precedes catalysis. The translocation step appears to follow a push-pull mechanism where the O-O1 loop of the finger subdomain acts as a pawl to facilitate unidirectional movement along the template with conserved tyrosine residues 714 and 719 functioning as tandem gatekeepers of DNA synthesis. The structures capture the precise order of critical events that may be a general feature of enzymatic catalysis among replicative DNA polymerases.


Assuntos
DNA Polimerase I/metabolismo , Replicação do DNA , DNA Bacteriano/metabolismo , Proteínas de Escherichia coli/metabolismo , Cristalografia por Raios X , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Genéticos , Conformação de Ácido Nucleico , Fatores de Tempo
8.
PLoS One ; 16(4): e0249354, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33793664

RESUMO

Q fever is one of the most important zoonotic diseases caused by the obligate intracellular bacteria, Coxiella burnetii. This bacterial infection has been frequently reported in both humans and animals, especially ruminants. Ticks are important ectoparasite and serve as reservoir hosts of Coxiella-like endosymbionts (CLEs). In this study, we have attempted to express chaperone-coding genes from CLEs of Rhipicephalus annulatus ticks collected fromcow path. The partial DnaK coding sequence has been amplified and expressed by Escherichia coli. Amino acid sequences have been analyzed by MS-MS spectrometry and the UniProt database. Despites nucleotide sequences indicating high nucleotide variation and diversity, many nucleotide substitutions are synonymous. In addition, amino acid substitutions compensate for the physicochemical properties of the original amino acids. Immune Epitope Database and Analysis Resource (IEDB-AR) was employed to indicate the antigenicity of the partial DnaK protein and predict the epitopes of B-and T-cells. Interestingly, some predicted HLA-A and B alleles of the MHC-I and HLA-DR alleles belonging to MHC-II were similar to T-cell responses to C. burnetii in Q fever patients. Therefore, the partial DnaK protein of CLE from R. annulatus could be considered a vaccine candidate and immunogenic marker with future prospects.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Coxiella burnetii/metabolismo , Rhipicephalus/microbiologia , Adenosina Trifosfatases/classificação , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Coxiella burnetii/isolamento & purificação , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Bases de Dados Genéticas , Epitopos/análise , Epitopos/imunologia , Haplótipos , Mutação , Filogenia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Simbiose
9.
Nat Chem Biol ; 17(6): 724-731, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33820990

RESUMO

Genetically modified microorganisms (GMMs) can enable a wide range of important applications including environmental sensing and responsive engineered living materials. However, containment of GMMs to prevent environmental escape and satisfy regulatory requirements is a bottleneck for real-world use. While current biochemical strategies restrict unwanted growth of GMMs in the environment, there is a need for deployable physical containment technologies to achieve redundant, multi-layered and robust containment. We developed a hydrogel-based encapsulation system that incorporates a biocompatible multilayer tough shell and an alginate-based core. This deployable physical containment strategy (DEPCOS) allows no detectable GMM escape, bacteria to be protected against environmental insults including antibiotics and low pH, controllable lifespan and easy retrieval of genomically recoded bacteria. To highlight the versatility of DEPCOS, we demonstrated that robustly encapsulated cells can execute useful functions, including performing cell-cell communication with other encapsulated bacteria and sensing heavy metals in water samples from the Charles River.


Assuntos
Bactérias/efeitos dos fármacos , Hidrogéis/farmacologia , Alginatos/química , Antibacterianos/farmacologia , Bactérias/genética , Materiais Biocompatíveis , Bioengenharia , DNA Bacteriano/química , DNA Bacteriano/genética , Monitoramento Ambiental , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Heme/química , Metais Pesados/química , Organismos Geneticamente Modificados , Percepção de Quorum , Rios , Poluentes da Água/química
10.
PLoS Comput Biol ; 17(4): e1008869, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33861734

RESUMO

ParABS, the most widespread bacterial DNA segregation system, is composed of a centromeric sequence, parS, and two proteins, the ParA ATPase and the ParB DNA binding proteins. Hundreds of ParB proteins assemble dynamically to form nucleoprotein parS-anchored complexes that serve as substrates for ParA molecules to catalyze positioning and segregation events. The exact nature of this ParBS complex has remained elusive, what we address here by revisiting the Stochastic Binding model (SBM) introduced to explain the non-specific binding profile of ParB in the vicinity of parS. In the SBM, DNA loops stochastically bring loci inside a sharp cluster of ParB. However, previous SBM versions did not include the negative supercoiling of bacterial DNA, leading to use unphysically small DNA persistences to explain the ParB binding profiles. In addition, recent super-resolution microscopy experiments have revealed a ParB cluster that is significantly smaller than previous estimations and suggest that it results from a liquid-liquid like phase separation. Here, by simulating the folding of long (≥ 30 kb) supercoiled DNA molecules calibrated with realistic DNA parameters and by considering different possibilities for the physics of the ParB cluster assembly, we show that the SBM can quantitatively explain the ChIP-seq ParB binding profiles without any fitting parameter, aside from the supercoiling density of DNA, which, remarkably, is in accord with independent measurements. We also predict that ParB assembly results from a non-equilibrium, stationary balance between an influx of produced proteins and an outflux of excess proteins, i.e., ParB clusters behave like liquid-like protein condensates with unconventional "leaky" boundaries.


Assuntos
Proteínas de Bactérias/química , Centrômero/química , Segregação de Cromossomos , DNA Bacteriano/química , DNA Super-Helicoidal/química , Modelos Biológicos , Nucleoproteínas/química , Ligação Proteica , Processos Estocásticos
11.
Arch Microbiol ; 203(6): 3279-3285, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33860341

RESUMO

A halophilic, Gram-staining-negative, rod-shaped, flagellated and motile bacterium, strain QX-1 T, was isolated from deep-sea sediment at a depth of 3332 m in the southwestern Indian Ocean. Strain QX-1 T growth was observed at 4-50 °C (optimum 37 °C), pH 5.0-11.0 (optimum pH 7.0), 3-25% NaCl (w/v; optimum 7%), and it did not grow without NaCl. A phylogenetic analysis based on the 16S rRNA gene placed strain QX-1 T in the genus Halomonas and most closely related to Halomonas sulfidaeris (97.9%), Halomonas zhaodongensis (97.8%), Halomonas songnenensis (97.6%), Halomonas hydrothermalis (97.4%), Halomonas subterranea (97.3%), Halomonas salicampi (97.1%), and Halomonas arcis (97.0%). DNA-DNA hybridization (< 26.5%) and average nucleotide identity values (< 83.5%) between strain QX-1 T and the related type strains meet the accepted criteria for a new species. The principal fatty acids (> 10%) of strain QX-1 T are C16:0 (25.5%), C17:0 cyclo (14.0%), C19:0 cyclo ω8c (18.7%), and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c, 18.1%). The polar lipids of strain QX-1 T are mainly diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, unidentified phospholipid, unidentified aminophospholipid, and five unidentified lipids. The main respiratory quinone is Q-9. The G + C content of its chromosomal DNA is 54.4 mol%. Its fatty acid profile, respiratory quinones, and G + C content also support the placement of QX-1 T in the genus Halomonas. These phylogenetic, phenotypic, and chemotaxonomic analyses indicate that QX-1 T is a novel species, for which the name Halomonas maris is proposed. The type strain is QX-1 T (= MCCC 1A17875T = KCTC 82198 T = NBRC 114670 T).


Assuntos
Sedimentos Geológicos/microbiologia , Halomonas/isolamento & purificação , Composição de Bases , DNA Bacteriano/química , Ácidos Graxos/análise , Halomonas/química , Halomonas/classificação , Halomonas/genética , Oceano Índico , Lipídeos/análise , Filogenia , Tolerância ao Sal
12.
Arch Microbiol ; 203(6): 3287-3294, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33860851

RESUMO

A Gram-positive, aerobic, non-motile, non-spore-forming, short rod-shaped strain, NEAU-LLCT, was isolated from cow dung in Shangzhi City, Heilongjiang Province, Northeast China and identified by a polyphasic taxonomic study. Colonies was light yellow, round, with entire margin. Strain NEAU-LLCT was grown at 15-45 â„ƒ and pH 6.0-10.0. NaCl concentration ranged from 0 to 5% (W/V). The 16S rRNA gene sequence of NEAU-LLCT showed the high similarities with Microbacterium kyungheense JCM 18735T (98.5%), Microbacterium trichothecenolyticum JCM 1358T (98.3%) and Microbacterium jejuense JCM 18734T (98.2%). The whole-cell sugars were glucose, rhamnose and ribose. The menaquinones contained MK-12 and MK-13. Ornithine, glutamic acid, lysine and a small amount of alanine and glycine were the amino acids in the hydrolyzed products of the cell wall. The major fatty acids were iso-C16:0, iso-C18:0, anteiso-C15:0 and anteiso-C17:0. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and an unidentified glycolipid. The genome of NEAU-LLCT was 4,369,375 bp and G + C content is 70.28 mol%. A combination of DNA-DNA hybridization result and some phenotypic characteristics demonstrated that strain NEAU-LLCT could be distinguished from its closely related strains. Therefore, the strain NEAU-LLCT was considered to represent a novel species, which was named Microbacterium helvum sp. (Type strain NEAU-LLCT = CCTCC AA 2018026T = JCM 32661T).


Assuntos
Microbacterium/isolamento & purificação , Aminoácidos/análise , Animais , Composição de Bases , Bovinos , DNA Bacteriano/química , Ácidos Graxos/química , Fezes/microbiologia , Feminino , Lipídeos/análise , Microbacterium/química , Microbacterium/classificação , Microbacterium/genética , Filogenia , Açúcares/análise
13.
Arch Microbiol ; 203(6): 3373-3388, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33880605

RESUMO

Mitsuaria sp. TWR114 is a biocontrol agent against tomato bacterial wilt (TBW). We aimed to gain genomic insights relevant to the biocontrol mechanisms and colonization ability of this strain. The draft genome size was found to be 5,632,523 bp, with a GC content of 69.5%, assembled into 1144 scaffolds. Genome annotation predicted a total of 4675 protein coding sequences (CDSs), 914 pseudogenes, 49 transfer RNAs, 3 noncoding RNAs, and 2 ribosomal RNAs. Genome analysis identified multiple CDSs associated with various pathways for the metabolism and transport of amino acids and carbohydrates, motility and chemotactic capacities, protection against stresses (oxidative, antibiotic, and phage), production of secondary metabolites, peptidases, quorum-quenching enzymes, and indole-3-acetic acid, as well as protein secretion systems and their related appendages. The genome resource will extend our understanding of the genomic features related to TWR114's biocontrol and colonization abilities and facilitate its development as a new biopesticide against TBW.


Assuntos
Agentes de Controle Biológico , Burkholderiales/genética , Genoma Bacteriano , Lycopersicon esculentum/microbiologia , Doenças das Plantas/prevenção & controle , Proteínas de Bactérias/genética , Composição de Bases , Agentes de Controle Biológico/metabolismo , Burkholderiales/metabolismo , DNA Bacteriano/química , Genômica , Ácidos Indolacéticos/metabolismo , Doenças das Plantas/microbiologia , Metabolismo Secundário/genética , Estresse Fisiológico
14.
Int J Mol Sci ; 22(8)2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923762

RESUMO

The efficiency of existing cell lysis methods to isolate nucleic acids from diverse bacteria varies depending on cell wall structures. This study tested a novel idea of using broad-spectrum antimicrobial peptides to improve the lytic efficiency of hard-to-lyse bacteria and characterized their differences. The lysis conditions of Staphylococcus aureus using recombinant porcine myeloid antimicrobial peptide 36 (PMAP-36), a broad-spectrum pig cathelicidin, was optimized, and RNA isolation was performed with cultured pellets of ten bacterial species using various membranolytic proteins. Additionally, three other antimicrobial peptides, protegrin-1 (PG-1), melittin, and nisin, were evaluated for their suitability as the membranolytic agents of bacteria. However, PMAP-36 use resulted in the most successful outcomes in RNA isolation from diverse bacterial species. The amount of total RNA obtained using PMAP-36 increased by ~2-fold compared to lysozyme in Salmonella typhimurium. Streptococci species were refractory to all lytic proteins tested, although the RNA yield from PMAP-36 treatment was slightly higher than that from other methods. PMAP-36 use produced high-quality RNA, and reverse transcription PCR showed the efficient amplification of the 16S rRNA gene from all tested strains. Additionally, the results of genomic DNA isolation were similar to those of RNA isolation. Thus, our findings present an additional option for high quality and unbiased nucleic acid isolation from microbiomes or challenging bacterial strains.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , RNA Bacteriano/química , Staphylococcus aureus/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Fracionamento Celular/métodos , Fracionamento Celular/normas , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , RNA Bacteriano/isolamento & purificação , Staphylococcus aureus/efeitos dos fármacos
15.
BMC Infect Dis ; 21(1): 352, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33858378

RESUMO

BACKGROUND: Identifying the causes of community-acquired pneumonia (CAP) is challenging due to the disease's complex etiology and the limitations of traditional microbiological diagnostic methods. Recent advances in next generation sequencing (NGS)-based metagenomics allow pan-pathogen detection in a single assay, and may have significant advantages over culture-based techniques. RESULTS: We conducted a cohort study of 159 CAP patients to assess the diagnostic performance of a clinical metagenomics assay and its impact on clinical management and patient outcomes. When compared to other techniques, clinical metagenomics detected more pathogens in more CAP cases, and identified a substantial number of polymicrobial infections. Moreover, metagenomics results led to changes in or confirmation of clinical management in 35 of 59 cases; these 35 cases also had significantly improved patient outcomes. CONCLUSIONS: Clinical metagenomics could be a valuable tool for the diagnosis and treatment of CAP. TRIAL REGISTRATION: Trial registration number with the Chinese Clinical Trial Registry: ChiCTR2100043628 .


Assuntos
Infecções Comunitárias Adquiridas/diagnóstico , Metagenômica/métodos , Pneumonia/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Líquido da Lavagem Broncoalveolar/microbiologia , Estudos de Coortes , Infecções Comunitárias Adquiridas/microbiologia , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Pneumonia/microbiologia , Análise de Sequência de DNA , Escarro/microbiologia , Adulto Jovem
16.
PLoS One ; 16(3): e0242396, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33720954

RESUMO

The objective of this pilot study was to describe the microbial profiles present in the plaque and saliva of children who continued to develop new carious lesions following treatment with silver diamine fluoride ("nonresponders") compared to caries active, caries-free, and children immediately receiving SDF treatment for untreated caries in order to identify potential microbial differences that may relate to a re-incidence of caries. Saliva and plaque samples from infected and contralateral sites were obtained from twenty children who were either caries free, had active carious lesions, were caries active and received SDF treatment immediately before sampling, or had previously received SDF treatment and developed new caries. In total, 8,057,899 Illumina-generated sequence reads from 60 samples were obtained. Reads were processed using the Quantitative Insights Into Microbial Ecology pipeline. Group differences were assessed using Analysis of Variance Models and Tukey Honest Significant Differences. To identify significant taxa between treatment groups, Linear discriminant analysis Effect Size (LefSe) and Analysis of Differential Abundance Taking Sample Variation Into Account were used. Differential abundant analysis indicated that members of the Lachnospiraceae family were significantly enriched in non-responders and the genus Tannerella and species Granulicatella adiances were also highly abundant in this group. LefSe analysis between non-responders and SDF-treated groups revealed that genera Leptotrichia and Granulicatella were enriched in non-responders. We observed the highest abundance of phosphotransferase system and lowest abundance of lipopolysaccharide synthesis in non-responders. The microbiome in dental biofilms is responsible for initiation and progression of dental caries. SDF has been shown to be effective in arresting the progression carious lesions, in part due to its antimicrobial properties. Findings suggest that the differential abundance of select microbiota and specific pathway functioning in individuals that present with recurrent decay after SDF treatment may contribute to a potential failure of silver diamine fluoride to arrest dental caries. However, the short duration of sample collection following SDF application and the small sample size emphasize the need for further data and additional analysis.


Assuntos
Cárie Dentária/tratamento farmacológico , Microbiota , Compostos de Amônio Quaternário/uso terapêutico , Compostos de Prata/uso terapêutico , Carnobacteriaceae/genética , Carnobacteriaceae/isolamento & purificação , Criança , Estudos Transversais , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Cárie Dentária/patologia , Placa Dentária/microbiologia , Análise Discriminante , Fluoretos Tópicos/uso terapêutico , Humanos , Leptotrichia/genética , Leptotrichia/isolamento & purificação , Projetos Piloto , Análise de Componente Principal , Saliva/microbiologia , Análise de Sequência de DNA , Falha de Tratamento
17.
PLoS One ; 16(3): e0242294, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33761524

RESUMO

Whole genome sequencing (WGS) provides essential public health information and is used worldwide for pathogen surveillance, epidemiology, and source tracking. Foodborne pathogens are often sequenced using rapid library preparation chemistries based on transposon technology; however, this method may miss random segments of genomes that can be important for accurate downstream analyses. As new technologies become available, it may become possible to achieve better overall coverage. Here we compare the sequence quality obtained using libraries prepared from the Nextera XT and Nextera DNA Prep (Illumina, San Diego, CA) chemistries for 31 Shiga toxin-producing Escherichia coli (STEC) O121:H19 strains, which had been isolated from flour during a 2016 outbreak. The Nextera DNA Prep gave superior performance metrics including sequence quality, assembly quality, uniformity of genome coverage, and virulence gene identification, among other metrics. Comprehensive detection of virulence genes is essential for making educated assessments of STECs virulence potential. The phylogenetic SNP analysis did not show any differences in the variants detected by either library preparation method which allows isolates prepared from either library method to be analysed together. Our comprehensive comparison of these chemistries should assist researchers wishing to improve their sequencing workflow for STECs and other genomic risk assessments.


Assuntos
Genoma Bacteriano , Escherichia coli Shiga Toxigênica/genética , Virulência/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Biblioteca Gênica , Tipagem de Sequências Multilocus , Filogenia , Polimorfismo de Nucleotídeo Único , Sorogrupo , Escherichia coli Shiga Toxigênica/classificação , Escherichia coli Shiga Toxigênica/patogenicidade , Sequenciamento Completo do Genoma
18.
Bioelectrochemistry ; 140: 107801, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33789176

RESUMO

Alicyclobacillus acidoterrestris is an acidophilic and thermophilic bacterium present in the soil, often associated with the spoilage of acidic juices, such as orange juice. Their spores resist pasteurization and, when reactivated, modify the organoleptic properties of the juice, making it unsuitable for consumption, due mainly to production of guaiacol. Biosensors are detection devices that respond quickly and are easy to handle, with great potential for use in the juice production chain. In this context, this work reports an electrochemical genosensor for detection of A. acidoterrestris, based on a graphite electrode modified with electrochemically reduced graphene oxide, a polymer derived from 3-hydroxybenzoic acid and a specific DNA probe sequence complementary with the genomic DNA of A. acidoterrestris. Detection of the target was performed by monitoring the oxidation peak of the Hoechst 33258, a common DNA stainer. The genosensor detection limit was 12 ng mL-1 and it kept 77% of response after ten weeks, and a test showed that orange juice does not interfere with bacteria lysate detection. This biosensor is the first platform for electrochemical detection of the genomic DNA of A. acidoterrestris in the literature, and the first to use Hoechst 33258 as indicator with whole genomic DNA molecules.


Assuntos
Alicyclobacillus/isolamento & purificação , Técnicas Biossensoriais/métodos , Bisbenzimidazol/química , DNA Bacteriano/análise , DNA Bacteriano/química , Alicyclobacillus/genética , Eletroquímica , Eletrodos , Grafite/química , Oxirredução
19.
Proc Natl Acad Sci U S A ; 118(10)2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33649196

RESUMO

We study the effect of transcription on the kinetics of DNA supercoiling in three dimensions by means of Brownian dynamics simulations of a single-nucleotide-resolution coarse-grained model for double-stranded DNA. By explicitly accounting for the action of a transcribing RNA polymerase (RNAP), we characterize the geometry and nonequilibrium dynamics of the ensuing twin supercoiling domains. Contrary to the typical textbook picture, we find that the generation of twist by RNAP results in the formation of plectonemes (writhed DNA) some distance away. We further demonstrate that this translates into an "action at a distance" on DNA-binding proteins; for instance, positive supercoils downstream of an elongating RNAP destabilize nucleosomes long before the transcriptional machinery reaches the histone octamer. We also analyze the relaxation dynamics of supercoiled double-stranded DNA, and characterize the widely different timescales of twist diffusion, which is a simple and fast process, and writhe relaxation, which is much slower and entails multiple steps.


Assuntos
Proteínas de Bactérias , DNA Bacteriano , DNA Super-Helicoidal , Proteínas de Ligação a DNA , RNA Polimerases Dirigidas por DNA , Transcrição Genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , DNA Bacteriano/química , DNA Bacteriano/metabolismo , DNA Super-Helicoidal/química , DNA Super-Helicoidal/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Simulação de Dinâmica Molecular
20.
Biochimie ; 185: 22-32, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33727139

RESUMO

Deinococcus radiodurans survives extraordinary doses of ionizing radiation and desiccation that cause numerous DNA strand breaks. D. radiodurans DNA polymerase A (DrPolA) is essential for reassembling the shattered genome, while its biochemical property has not been fully demonstrated. In this study, we systematically examined the enzymatic activities of DrPolA and characterized its unique features. DrPolA contains an N-terminal nuclease domain (DrPolA-NTD) and a C-terminal Klenow fragment (KlenDr). Compared with the Klenow fragment of E. coli Pol I, KlenDr shows higher fidelity despite the lacking of 3'-5' exonuclease proofreading activity and prefers double-strand DNA rather than Primer-Template substrates. Apart from the well-annotated 5'-3' exonuclease and flap endonuclease activities, DrPolA-NTD displays approximately 140-fold higher gap endonuclease activity than its homolog in E. coli and Human FEN1. Its 5'-3' exonuclease activity on ssDNA, gap endonuclease, and Holliday junction cleavage activities are greatly enhanced by Mn2+. The DrPolA-NTD deficient strain shows increased sensitivity to UV and gamma-ray radiation. Collectively, our results reveal distinct biochemical characteristics of DrPolA during DNA degradation and re-synthesis, which provide new insight into the outstanding DNA repair capacity of D. radiodurans.


Assuntos
Proteínas de Bactérias/química , DNA Polimerase III/química , DNA Bacteriano/química , Deinococcus/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Deinococcus/genética , Humanos
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